Optical Clearing and Labeling for Light-sheet Fluorescence Microscopy in Large-scale Human Brain Imaging

作者： Danila Di Meo*1, Josephine Ramazzotti*1, Marina Scardigli*1,2, Franco Cheli1, Luca Pesce1,3, Niamh Brady1, Giacomo Mazzamuto1,4,5, Irene Costantini1,4,6, Francesco S. Pavone1,4,5 1European Laboratory for Non-linear Spectroscopy (LENS),University of Florence, 2Division of Physiology, Department of Experimental and Clinical Medicine,University of Florence, 3Department of Physics,University of Pisa, 4National Research Council - National Institute of Optics (CNR-INO), 5Department of Physics and Astronomy,University of Florence, 6Department of Biology,University of Florence

简介：

Overview

This study presents a high-throughput protocol for simultaneous optical clearing, multi-round labeling, and 3D volumetric reconstruction of postmortem human brain sections. By integrating the S WITCH - H 2 O 2 - Antigen R etrieval - 2,2'-thiodiethanol (TDE) SHORT technique with light-sheet fluorescence microscopy, the methodology allows for detailed structural characterization at micrometer resolution.

Key Study Components

Area of Science

• Neuroscience
• Histology
• Immunohistochemistry

Background

• The human brain is a complex organ requiring intricate examination across varying scales.
• Challenges include dealing with large brain samples and autofluorescent signals from pigments.
• The presented protocol addresses shortcomings of existing clearing techniques.

Purpose of Study

• To develop a streamlined protocol for analyzing the structure of the human brain.
• To enhance optical clearing efficiency and imaging quality of brain tissue sections.
• To enable 3D reconstruction with high detail and reliability.

Methods Used

• The study employs light-sheet fluorescence microscopy.
• Human brain tissue is used as a biological model, with detailed sectioning and processing steps outlined.
• Key steps include embedding samples in agarose, processing with TDE, and multi-round immunostaining for various neuronal markers.
• Incubation times and temperatures are precisely defined to ensure optimal clearing and imaging conditions.

Main Results

• The protocol leads to effective optical clearing and high-quality imaging of gray and white matter.
• Multi-round staining allows visualization of distinct neuronal populations in human brain sections.
• The method demonstrates the ability to capture subcellular detail in 3D reconstructions.

Conclusions

• This study establishes a method for comprehensive analysis of brain structure at micrometer resolution.
• The approach highlights the potential for advancing our understanding of brain architecture and its implications for neuronal studies.

Frequently Asked Questions